The morphology of short actin filament interaction with the myosin I from Acanthamoeba was examined. Short actin filaments were produced by addition of gelsolin to actin in a ratio of 1:50. With an actin/myosin I molar ration of 10/1 slender bundles of actin formed and were stable in the absence of ATP. Actin filaments were held in parallel alignment with a spacing of about 69 nm. This was in contrast to controls, lacking myosin I, in which actin filaments often appear to be aligned with no space between them. The distance between two actin filaments in the bundles is compatible with a single myosin I molecule forming the cross-link. In two-dimensional arrays of cross-linked filaments the cross links appear to be spaced at 25-30 nm intervals along the filament. The three-dimensional appearance of actin (in the absence of gelsolin) and myosin I gels is that of an anastomosing network of long actin filaments that appear to be linked by association into bundles similar in apperance to those seen with short actin.